Recent advances in gene transfer have made possible the introduction of genetic material into intact animals or their bone marrow cells. One goal of these experiments is to insert altered dihydrofolate reductase (DHFR) genes into bone marrow, thereby rendering it resistant to methotrexate (MTX). This would allow treatment of malignancies with high doses of MTX without fear of bone marrow toxicity. If such a system worked it could be applied to human beings. The problem with experiments attempted thus far is that gene transfer protocols are not effective enough to assure that the bone marrow will be uniformly transformed with an efficiently expressed MTX-resistant DHFR gene. Thus, the applicability of this approach to humans remains to be demonstrated. The purpose of our experiments is to establish a system wherein the chances of inducing host resistance to MTX are optimized. We have produced a number of lines of transgenic mice by microinjecting a mutant DHFR gene into one-celled embryos. This gene specifies a product that is highly resistant to MTX. All animals have the new gene integrated into the germ line and transmit it to progeny, which now carry it in every cell. Several lines also express the gene in several somatic tissues including bone marrow. We will administer MTX to progeny and control littermates not carrying the DHFR gene in order to select in vivo for resistance to the drug. When these mice develop MTX resistance, we will cross them to inbred animals and innoculate the transgenic progeny with malignant cells. We will then give MTX to determine if cancer can be cured in the genetically MTX-resistant transgenic mice. We also propose to improve the model for isolated bone marrow resistance. Our approach entails the production of additional transgenic mice on the inbred C3H background. After the animals have been selected for bone marrow resistance we will transplant their bone marrow to irradiated C3H recipients. These latter animals will thus receive bone marrow cells which are uniformly transformed with the MTX-resistant DHFR gene and which are known to express the gene efficiently. The response of these animals to MTX therapy should provide definitive information regarding the feasibility of gene transfer as an adjunct to chemotherapy.